Safo Laudina, Abdelrazig Salah, Grosse-Honebrink Alexander, Millat Thomas, Henstra Anne M, Norman Rupert, Thomas Neil R, Winzer Klaus, Minton Nigel P, Kim Dong-Hyun, Barrett David A
Centre for Analytical Bioscience, Advanced Materials and Healthcare Technologies Division, School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, U.K.
Biodiscovery Institute, School of Chemistry, University of Nottingham, Nottingham NG7 2RD, U.K.
ACS Omega. 2021 May 20;6(21):13518-13526. doi: 10.1021/acsomega.0c05588. eCollection 2021 Jun 1.
We report a liquid chromatography-isotope dilution mass spectrometry method for the simultaneous quantification of 131 intracellular bacterial metabolites of . A comprehensive mixture of uniformly C-labeled internal standards (U-C IS) was biosynthesized from the closely related bacterium using 4% C-glucose as a carbon source. The U-C IS mixture combined with C authentic standards was used to validate the linearity, precision, accuracy, repeatability, limits of detection, and quantification for each metabolite. A robust-fitting algorithm was employed to reduce the weight of the outliers on the quantification data. The metabolite calibration curves were linear with ≥ 0.99, limits of detection were ≤1.0 μM, limits of quantification were ≤10 μM, and precision/accuracy was within RSDs of 15% for all metabolites. The method was subsequently applied for the daily monitoring of the intracellular metabolites of during a CO gas fermentation over 40 days as part of a study to optimize biofuel production. The concentrations of the metabolites were estimated at steady states of different pH levels using the robust-fitting mathematical approach, and we demonstrate improved accuracy of results compared to conventional regression. Metabolic pathway analysis showed that reactions of the incomplete (branched) tricarboxylic acid "cycle" were the most affected pathways associated with the pH shift in the bioreactor fermentation of and the concomitant changes in ethanol production.
我们报告了一种液相色谱 - 同位素稀释质谱法，用于同时定量[细菌名称]的131种细胞内细菌代谢物。使用4％的[碳源名称] - 葡萄糖作为碳源，从密切相关的细菌中生物合成了均匀的[碳标记方式] - C标记内标（U - C IS）的综合混合物。将U - C IS混合物与[碳标记方式] - C真实标准品结合，用于验证每种代谢物的线性、精密度、准确度、重复性、检测限和定量限。采用稳健拟合算法来减轻定量数据中异常值的权重。代谢物校准曲线呈线性，[相关系数要求]≥0.99，检测限≤1.0μM，定量限≤10μM，所有代谢物的精密度/准确度在相对标准偏差（RSD）的15％以内。作为优化生物燃料生产研究的一部分，该方法随后应用于在40天的[气体名称]CO气体发酵过程中对[细菌名称]细胞内代谢物的日常监测。使用稳健拟合数学方法在不同pH水平的稳态下估计代谢物的浓度，并且我们证明与传统回归相比结果的准确性有所提高。代谢途径分析表明，不完全（分支）三羧酸“循环”的反应是与[细菌名称]生物反应器发酵中的pH变化以及乙醇产量的伴随变化相关的最受影响的途径。
